The goal of this research effort is to understand how various types of white blood cells recognize and respond to the presence of a microorganism or cancer cell in the body, or inappropriately recognize a normal component of the body (an auto-antigen). Our experiments are designed to provide a detailed understanding of how the substances (antigens) making up microorganisms, cancer cells, or normal self-components, are made visible to the defending cells of the innate (anti-unspecific) or adaptive (antigen-specific) limbs of the host defense system and how recognition of infections, malignant cells, or adjuvants is linked through complex cell-cell interactions to the induction of protective or self-destruction effector responses. We are using our 2 photon intravital imaging technology on a range of projects investigating the intersection of the immune system with vaccines, infectious antigen sources, and dying cells in tissues. We are extending our earlier studies on the chemokine control of CD4-CD8 T cell interactions to better understand how these cell types collaborate in lymphoid tissue to generate optimal primary and memory cell-mediated immune responses. We previously found that different dendritic cells initially present antigen to the majority of CD4 vs. CD8 T cells. In FY14 we have used a combination of technologies to identify each antigen-presenting cell type and show that eventually, both CD4 and CD8 T cells locate the same CD8, XCR1+ dendritic cell, which serves as the platform by exchange of signals that augment the CD8 effector and/or memory response. We have also expanded our imaging technologies by developing methods that permit performing 7 or 8 color fluorescent immunohistochemistry on lymphoid and other tissues, collecting tiled images across entire tissue sections, and computationally analyzing the data to assign multiple stains to specific cells. This method has allowed us to fully characterize all the migratory and resident dendritic cell subpopulations in skin draining lymph nodes of mice and to reveal the complex and heterogeneous, but non-random distribution of these various dendritic cell subsets. It has also permitted us to generate hypotheses about which dendritic cell subsets capture antigen delivered in particular ways and to begin to test these models by direct imaging, in concert with an assessment of the T cell subsets with which such antigen-capturing dendritic cells interact. As part of this study, we have developed a new pipeline for image capture, processing, and analysis that permits not only the analysis of cell phenotype and positioning, but also a quantitative assessment of these phenotypically complex cell populations within intact tissue. We call this entire set of analytic tools Histo-cytometry for its similarity in outcome to flow cytometric analysis of dissociated cell populations. This methodology has potential applicability to human and NHP tissues and we have used this method in a collaborative study to examine follicular helper cell (Tfh) in infected monkeys, work that should contribute to the improvement of vaccines. In FY14, we combined data from Histo-cytometry with 2P intravital imaging to identify a novel population of lymphatic sinus-resident dendritic cells in mouse lymph nodes and to show that these cells extend cytoplasmic processes directly into the subcapsular sinus to sample material in draining lymph. These cells are uniquely able to acquire particulate antigens accessing the lymph node by this route, in comparison to dendritic cells localized deeper within the paracortex, with important implications for which dendritic cells present antigens delivered in particulate form. We also discovered that these sinus DC localize to the regions of the subcapsular sinus called interfollicular regions, where they replace the layer of CD169+ macrophages that are more restricted to the region over the primary B cell follicles, thus revealing a separation in antigen delivery function for T cells (sinus DCs) vs. B cells (sinus resident macrophages). Functional studies documented a key role of the sinus resident DC in promoting early T cell responses independent of migratory DC from the skin site of immunization, in contrast to existing dogma. In a new application of Histo-cytometry, we developed methods for combining cell phenotyping with analysis of phospho-proteins, especially pSTAT molecules. We focused on the latter to address the question of the range over which cytokines act in vivo. While screening a panel of anti-pSTAT reagents, we noted an unexpected pattern of staining with antibody to pSTAT5. Further analysis showed that these small clusters of pSTAT5+ cells in the peripheral regions of all lymph nodes were Foxp3+ Tregs, that the pSTAT5 signal came from a central CD4 Tiff cells making IL-2, that the clustered Tregs were especially rich in suppressive molecules like CTLA4 and CD73, that the TCR of Tregs was required for both the clustering phenomenon and effective suppression of Teff responses, and that IL-2 from the Teff was also required for optimal Treg suppression of responses. Most remarkably, the pSTAT5+ Treg clusters were of similar location and number on germfree mice. Together these data suggest that immune homeostatic is maintained not by Treg prevention of autoreactive T cell activation, but rather by a spatially localized feedback regulatory circuit in which activation of conventional T cells results in I-2 production that other with physical clustering and signaling mediated by the TCR of Tregs, results in abrogation of the developing effector T cell response. In FY14, using a novel method that permits the rapid screening of control vs. mutant (knock-out) immune cells for differences in behavior within a tissue site, we extended our studies on localized swarming response of neutrophils to sterile tissue damage. Studies using this method revealed a very specific role for the lipid mediator LTB4 in neutrophil responses to cell death as well as an unsuspected effect of neutrophil aggregation on local tissue matrix structure. Surprisingly, these imaging studies also revealed that neutrophils migrate in complex tissues in the apparent absence of a requirement of high affinity integrin function, though integrins play a role in creating a tight swarm of neutrophils that isolate damaged tissue. These studies are being extended further using 3D culture systems and microfabricated devices that permit precise control of chemoattractant gradient steepness and magnitude, as well as changes in effective concentration over time. Using these 3D systems, we have developed an entirely new picture of the factors controlling persistent migration of myeloid cells. Our new results suggest that temporal rather than spatial sensing plays a crucial role in persistent directional migration of various myeloid cell types to what are called guidance chemokines, results that fit with two sets of in vivo data that indicate robust amplification loops operating during innate inflammatory processes that would provide the needed rising chemoattractant gradients required for such directed migration in vivo.